The Clinical, Radiological and Genetic Spectrum of PLA2G6-Associated Neurodegeneration: An Experience From a Tertiary Center

Background: Despite being the second most common type of neurodegeneration with brain iron accumulation, there is limited literature on PLA2G6-associated neurodegeneration (PLAN) within the Asian ethnicity, particularly in the Indian context. Methods: We conducted a retrospective observational study on patients with pathogenic/likely pathogenic PLA2G6 variants based on exome sequencing. Results: We identified 26 patients (22 families, 15 males) of genetically-confirmed PLAN with a median age of 22.5 years and age at onset of 13.0 years, encompassing various subtypes: infantile neuroaxonal dystrophy (5/26;19.2%), atypical neuroaxonal dystrophy (3/26;11.5%), dystonia-parkinsonism (5/26;19.2%), dystonia-parkinsonism-myoclonus (n = 4, 15.38%), early-onset Parkinson’s disease (2/26;7.7%), complex dystonia (2/26;7.7%), and complicated hereditary spastic paraparesis (cHSP; 5/26;19.2%). The common initial symptoms included walking difficulty (7/26;26.9%), developmental regression (6/26;23.1%), and slowness (4/26;15.4%). Dystonia (14/26;53.8%), followed by parkinsonism (11/26; 42.3%), was the most common motor symptom. Non-motor symptoms included cognitive decline (12/26;46.2%) and behavioral changes (6/26;23.1%). Neuroimaging revealed cerebellar atrophy in 23/26 (88.5%) patients and claval hypertrophy in 80% (4/5) of INAD patients. Levodopa responsiveness was noted in 12 of 14 patients with parkinsonism/dystonia who received levodopa, and dyskinesia was noted in 10/11 patients. Genetic analysis revealed a total of 19 unique variants in PLA2G6 gene, of which 11 were novel. Twelve patients harbored the c.2222G>A variant, which is predominantly seen in Asian subpopulations. Conclusions: The study introduces 26 new patients of PLAN and 12 patients associated with the c.2222G>A variant, potentially forming the most extensive single center series to date. It also expands the phenotypic, neuroimaging, and genotypic spectrum of PLAN.


INTRODUCTION
PLA2G6-associated neurodegeneration (PLAN), representing the second most common type of neurodegeneration with brain iron accumulation (NBIA), encompasses a number of unique clinical entities [1].Depending on the clinical manifestation across different stages of life, these comprise infantile neuroaxonal dystrophy (INAD, NBIA2 A; MIM 256600), atypical neuroaxonal dystrophy (aNAD, NBIA2B; MIM 610217), and adult-onset dystoniaparkinsonism (DP, PARK14, MIM 612953) [1,2].Diagnosis and subcategorization into distinct subgroups depend on various factors such as clinical symptoms, disease progression, neurophysiological assessments, radiographic studies, and laboratory tests [3].Establishing a genetic diagnosis for these phenotypes constitutes a definite challenge owing to the complexities of clinical presentation and the rarity of this disease.At the molecular level, the PLA2G6 gene is situated on chromosome 22 at 22q13.1, and contains 17 exons spanning over 69 kilobases (kb).It encodes the CaI-PLA2 protein, which is pivotal in catalyzing hydrolysis within phospholipids.Mutations in PLA2G6 disrupt its function of repairing oxidative damage in phospholipid membranes, which affects the membrane integrity and fluidity, thereby contributing to the underlying pathological mechanisms.
Previous large-scale studies have continued to add to the constellation of phenotypes, highlighting the importance of having a high index suspicion despite atypical clinical presentation [1,3].As the disease continues to evolve, recent studies have revealed the role of ethnicity in dictating the genotypic variants and the clinical phenotype.Against this backdrop, we conducted a retrospective observational single-center study to describe in-depth, the clinical phenotypes and correlate them with the genotype of the patients evaluated so far in our neurology center.

PATIENTS AND METHODS
We performed a retrospective observational study of all patients exhibiting a biallelic pathogenic or likely pathogenic variants in the PLA2G6 gene based on exome sequencing in the probands.The case records were subjected to detailed data extraction comprising details of age at onset (AAO), age at presentation, first symptom at onset, the spectrum of motor symptoms, movement disorders encountered, non-motor clinical features including cognitive, psychiatric, and additional relevant clinical features, genotype, management strategies including levodopa responsiveness and drug-induced dyskinesia.All patients were classified based on the clinical phenotype into INAD, aNAD, dystonia-parkinsonism (DP), dystonia parkinsonism myoclonus (DPM), early-onset parkinsonism (EOP), complex dystonia and complicated hereditary spastic paraparesis (cHSP) phenotype.The study was approved by the institute's ethics committee.Written informed consent was obtained from patients for the publication of recorded videos.

GENETIC ANALYSES
The blood samples of patients were subjected to Genomic DNA extraction using QIAamp DNA Blood Mini Kit (Qiagen Germany, #51104).Subsequently, raw reads were aligned to the human reference genome (GRCh37) based on the BMA-mem algorithm.Polymerase chain reaction (PCR) duplicates were removed using the Picard toolkit (https:// broadinstitute.github.io/picard/)[4].The variants were identified based on the Genome Analysis Toolkit (GATK) framework (Broad Institute, Cambridge, MA, USA).Variants were exposed to base quality score recalibration for filtration, following which annotations would be done in the ANNOVAR platform (http://www.openbioinformatics. org/annovar/) [5].The variants with a minor allele frequency (MAF) >0.01, suggestive of common occurrence in the population, were not included.A comparison of data with the Exome Aggregation Consortium (ExAC), 1000 Genome project, and gnomAD database (https:// gnomad.broadinstitute.org/)was performed.Each of the individual sequence variants were tested using PolyPhen-2, Sorting Intolerant from Tolerant (SIFT) webserver, and MutationTaster [6][7][8].The variants were assigned a pathogenicity coding according to the American College of Medical Genetics and Genomics (ACMG) guidelines into benign, likely pathogenic or pathogenic [9].Each of these genes was analyzed in the mutation databases of ClinVar for novelty evaluation.

Conclusions:
The study introduces 26 new patients of PLAN and 12 patients associated with the c.2222G>A variant, potentially forming the most extensive single center series to date.It also expands the phenotypic, neuroimaging, and genotypic spectrum of PLAN.

REVIEW OF LITERATURE
We conducted a systematic search across the accessible medical database of PubMed, Google Scholar, and Scopus, using the medical subject headings (MeSH): "PLA2G6", "PLAN", "INAD", "ANAD", "atypical NAD", "Hereditary Spastic Paraparesis", parkinsonism", "young onset Parkinson's disease", "early onset Parkinson's disease", "dystonia", and "disorder" to identify all the relevant studies.The articles were subjected to title and abstract screening.A shadow search of the reference articles was done to avoid missing key articles.Our data extraction process was based on identifying unique demographic and clinical parameters, classifying the clinical phenotype, and describing the specific genetic variants so identified.Studies not presented in English or lacking patient details were excluded from consideration.

STATISTICAL METHODS
Categorical variables were depicted in terms of frequencies, whereas continuous variables were designated as median values along with their respective interquartile ranges.The initial analysis involved descriptive statistics to examine the demographic, clinical, and neuroimaging parameters.A narrative description encompassing clinical phenotypes and genotypes and their correlation with neuroimaging parameters was made.Statistical calculations were performed using SPSS version 23.0.

DEMOGRAPHICS
In this retrospective analysis, we analyzed 26 patients (22 families) with genetically confirmed PLAN.Eight of these patients were part of a multicentric publication [3].There was a male predominance (n = 15, 57.69%) distributed across 22 families.The median age at presentation was 22.5 years (IQR: 6.5-29.0),while the median age at onset (AAO) was 13.0 years (IQR: 2.7-20.5)and the patients presented after a median duration of illness of 3.5 months (IQR: 1.5-10.5).Consanguinity was observed in 53.8% of patients (n = 14), while 42.3% (n = 11) had a positive family history (Table 1).

CLINICAL FEATURES Symptomatology
At the onset of the disease, patients exhibited diverse symptoms, with difficulty in walking (7/

Other neurological signs
Optic atrophy was found in one (1/26;3.8%),and nystagmus was present in 3 patients (3/26;11.5%).Gaze restriction was a prevalent symptom, affecting 11 patients (11/26;42.3%).In most patients, the upgaze was mildly restricted, and there were no square wave jerks.Vestibulo-ocular maneuverer and optokinetic nystagmus reflex could not be performed in many.Vestibuloocular maneuverer was performed in two patients with cHSP phenotype, and the upgaze movement was better, suggesting a possibility of supranuclear type of abnormality.Twelve patients (12/

INVESTIGATIONS AND MANAGEMENT Neuroimaging features
Brain MRI (available for 25 patients) showed mineralization in various areas, including the caudate in 9/25 patients (34.6%), the putamen in 8/25 patients (30.8%), the GPi in 14/25 patients (53.8%), and the substantia nigra in With regards to the individual subtypes, classic claval hypertrophy was observed in 80% (n = 4/5) of patients of INAD followed by ANAD (1/3;33.3%) and DP (2/5;40%).This claval hypertrophy refers to the hypertrophy of the gracile tubercle formed by the nucleus and fasciculus of the gracilis due to spheroid bodies deposition in sensory nuclei of the medulla [10].The predominant MRI abnormalities were diffuse involvement of white matter (4/5;80%) in the DP subtype.Mineralization of substantia nigra and putamen was noted in a single patient with an EOP phenotype.The two patients with complex dystonia lacked any signature MRI involvement except for non-specific cerebellar atrophy (Figure 1, Table 1).

Management
A levodopa/carbidopa combination was administered in 14 patients with parkinsonism and/or dystonia.Twelve patients had at least 33% improvement either subjectively or objectively.Dopa-induced choreiform dyskinesia was noted in 10 out of 11 patients.Other symptomatic medications were administered, such as baclofen, clonazepam, tetrabenazine and trihexyphenidyl.None of the patients underwent deep brain stimulation (DBS) surgery (Table 1).

Genetic analysis
Exome sequencing (Figure 2, Table 2) revealed biallelic disease-causing variants in the PLA2G6 gene in all patients in homozygous state in 21 (21/26;80.8%)and In five patients with compound heterozygous variants, three patients had missense/missense configuration and one patient each had missense/stop-gain and missense/ frameshift configuration.The c.2222G>A;p.Arg741Gln variant was the most commonly identified variant in our cohort, which was identified in 12 patients (9 families).This variant has been previously reported and is predominantly seen in Asian subpopulations [1].The next common variant was a novel c.2405T>C;p.Leu802Pro missense variant, which was observed in two patients.It was identified in homozygous state in one patient with EOP and in compound heterozygous configuration with a previously reported pathogenic stop-gain variant in another patient with cHSP phenotype.In total 19 unique variants were identified, of which 15 were missense variants, two were stop-gain and the remaining two were frameshift variants (Figure 2).Among these 19 unique variants, 11 variants were novel.Of these 11 novel variants, 9 were missense variants (c.292T>C;p.Ser98Pro, c.379G>A;p.Val127Met, c.667C>A;p.Pro223Thr, c.763C>T;p.Pro255Ser, c.1471C>T;p.Leu491Phe, c.1763T>C;p.Leu588Pro, c.1897G>A,p.Ala633Thr, c.2197G>C;p.Asp733His and c.2405T>C;p.Leu802Pro) and two were frameshift variants (c.835delA;p.Ile279SerfsTer26 and c.1723delT;p.Thr575 ArgfsTer8).Of these, as per ACMG criteria, eight variants could be classified as pathogenic or likely pathogenic, while three variants (c.292T>C;p.Ser98Pro, c.379G>A;p.Val127Met and c.763C>T;p.Pro255Ser) could be only classified as a variant of uncertain significance (VUS) at best.However, as per the recent scoring update to ACMG criteria, these variants could be reclassified as VUS more likely to be pathogenic than benign (VUS-LP) [9].One novel variant (c.2405C>T;p.Leu802Pro) was seen in two patients, in homozygous state in a patient with EOP and in compound heterozygous trans configuration with a previously reported pathogenic stopgain variant in a patient with cHSP owing to which it could be classified to likely pathogenic variant.

CASE VIGNETTES
Patient-13: A 26-year-old previously healthy female with consanguineous parentage, normal birth and developmental history presented with a complex neurological illness spanning 10 years (Video-1).Initially,   she noticed walking difficulty with stiffness in limbs and occasional toe walking.In addition, she had frequent falls and inappropriate smiling.Her elder brother also had walking difficulty for 12 years duration, along with inappropriate smiling.On examination (Video-1), both patients had mild upgaze restriction that improved on vestibulo-ocular maneuverer, pseudobulbar affect, spasticity, and brisk deep tendon reflexes in all four limbs with extensor plantar response bilaterally and cerebellar abnormality.MRI of the proband revealed mineralization of basal ganglia and substantia nigra with cerebellar and cortical atrophy.Exome sequencing revealed a homozygous pathogenic missense variant (c.2222G>A;p.Arg741Gln).
The siblings were treated symptomatically with baclofen and physiotherapy.Patient-7: A 24-year-old female born to consanguineous parentage with normal birth and developmental history presented with a twelve-year history of progressive cognitive impairment, followed by tremulousness in upper limbs, slowness, abnormal posturing and walking difficulty.In addition, the patient had a behavioral abnormality in the form of anger outbursts, crying spells, low mood and anxiety.On examination (video-2), the proband had parkinsonism, rest tremor, facial predominant myoclonus, pyramidal signs, generalized dystonia and cerebellar involvement.MRI showed diffuse cortical and cerebellar atrophy with basal ganglia mineralization.Exome sequencing revealed a previously reported pathogenic homozygous missense variant (c.2222G>A;p.Arg741Gln).She was treated with levodopa with a good response but developed disabling choreiform dyskinesia.Her elder brother also had similar cognitive, behavioral, and motor symptoms of 12 years duration and, on examination, had parkinsonism, rest tremor, dystonia, pyramidal signs, myoclonus, and cerebellar abnormality.The rest tremor persisted in an outstretched position as well albeit with similar severity, and in the presence of cerebellar signs and distal hand dystonia, it can be a component of rubral tremor rather than parkinsonian rest tremor.Sanger sequencing in the brother confirmed the presence of c.2222G>A;p.Arg741Gln in homozygous state.He also responded to levodopa but with disabling dyskinesia.
Patient-25: A 20-year-old male born to consanguineous parentage with normal birth and developmental history presented with an eight-year history of head and right upper limb posturing and tremulousness, alongside intellectual disability (Video-3).He demonstrated upgaze restriction, ataxic dysarthria, and generalized dystonia, with normal gait and brisk reflexes.MRI also revealed cerebellar atrophy.Exome sequencing revealed a previously reported pathogenic homozygous missense variant (c.2222G>A;p.Arg741Gln).The neck dystonia was associated with axial paroxysms of jerky dystonia commonly seen in patients with myoclonus dystonia (DYT-SGCE).However, there were no significant variant identified in SGCE gene.He did not respond to levodopa therapy and was put on clonazepam and baclofen for dystonia.His elder sibling, 22-year-old girl also had similar symptoms but of only 4 months duration.Examination revealed upgaze restriction, generalized dystonia, appendicular ataxia, and brisk reflexes, with a dystonic
With regards to neuroimaging, cerebellar atrophy and mineralization in substantia nigra were seen in all patients with available brain MRI (n = 4).There were 3 patients (75%) that were detected to have mineralization involving caudate, putamen, and globus pallidus interna (GPi).Unlike previous reports, white matter signal changes were reported in 75% of patients (n = 3/4), while claval hypertrophy was reported in 2 patients.Interestingly, the most common genetic variant: c.2222G>A identified in 3 patients (60%), was not previously reported with PLA2G6related complicated cHSP phenotype.The difference in clinical phenotypes and neuroimaging findings as compared to the previous patients could be accounted for by the differences in the genotypic spectrum of the identified variants in the PLA2G6 gene.

PLA2G6-ASSOCIATED PHENOTYPES RELATED TO C.2222G>A VARIANT
The c.2222G>A is the most common genetic variant identified in our cohort of PLAN (34.6%).Previously 19 patients have been reported in the literature on this genetic variant (Table 5) [1,2,[16][17][18][19][20][21][22].Amongst the patients reported previously, a large proportion of them belong to Asian ancestry, and of these, 12 patients either are from India or have an Indian ancestry [1,[17][18][19][20][21].Our study contributes to the largest single-center series of patients identified with c.2222G>A variant.The demographic parameters of this subgroup were at par with previous publications, exhibiting a nearly equal sex ratio, similar AAO (Median: 28 years; IQR: 23.5-33 years), a similar prevalence of family history (77.8% vs 78.9%), and consanguinity (66.7% vs 72.2%).The unique attributes that were recognized include the occurrence of cHSP phenotype (n = 3, 33.3%),DPM (n = 3, 33.3%), and aNAD (n = 1, 11.1%), which were not known previously.While previous studies have suggested a much greater prevalence of DP (n = 16/19, 84.6%),only 2 of our patients (22.2%) displayed such a phenotype.A recent report demonstrated a remarkable response to bilateral subthalamic nuclei DBS in a patient diagnosed with PLAN carrying this specific variant in the homozygous state [21].At three months follow-up, the patient experienced significant relief from dyskinesia (>90%), improvement in OFF-state (UPDRS-III score 61 to 16), and reduction in levodopa-equivalent daily dose (1050 mg to 275 mg).This emphasizes the crucial importance of identifying this variant in patients clinically suspected of having this condition [1].However, the long-term sustenance of the benefit needs to be studied.There are two more reports of STN-DBS in patients with PLAN but with different variants.In the report by Wirth [24].However, no further details are available.In view of only a few case reports, one needs to be cautious with respect to the long-term benefits of STN-DBS in patients with PLAN.

LIMITATION
Our study has several limitations.Owing to the retrospective nature, the investigations are not uniform, and there is a possibility of missing clinical information.A formal cognitive assessment of patients with cognitive decline was not done in our patients.Additionally, a dedicated severity scale for parkinsonism and dystonia was not uniformly available.Previous studies have demonstrated the utility of tissue diagnosis that may aid in identifying axonal spheroids.However, none of our patients had any tissue diagnosis.The absence of longitudinal follow-up was one of the drawbacks.
to cHSP associated with PLAN.We observed three patients displaying a cHSP phenotype in patients with c.2222G>A variant.Furthermore, our study revealed 11 novel variants in the PLA2G6 gene, expressed both in homozygous and compound heterozygous states.

ETHICS AND CONSENT
Institute Ethics Committee approval was obtained for the study and informed consent was obtained from the patients for video recording and publishing.We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.

Figure 2 Video 1
Figure 2 Overview of the variants identified in this cohort.Image depicting the location of the identified variants in the PLA2G6 gene (Transcript ID: NM_003560.4).Novel variants are in italics, pathogenic variants are in red, likely pathogenic variants are in black and variant of uncertain significance are in blue.

Video 3
Video of Patient-25 with complex dystonia.Video of Patient-25 demonstrating cervical predominant generalized dystonia with left torticollis with frequent spasmodic retrocollis, mild dystonia in outstretched hands with dystonic right thumb tremor, and truncal tilt to right on walking.In addition, the patient had mild cerebellar signs and brisk deep tendon reflexes.Exome sequencing revealed homozygous c.2222G>A;p.Arg741Gln pathogenic missense variant.The video was taken after written informed consent was obtained for video recording, and publication in print and online.

Video 2
Video of Patient-7 with dystonia-parkinsonismmyoclonus phenotype.Video of Patient-7 demonstrating reduced facial expression, left more than right rest tremor, and distal appendicular predominant generalized dystonia.Post-levodopa, the patient developed generalized choreiform dyskinesia with partial improvement in parkinsonism and dystonia.In addition, the patient had facial predominant perioral action-induced myoclonus and pyramidal signs (not shown in the video).Exome sequencing revealed homozygous c.2222G>A;p.Arg741Gln pathogenic missense variant.The video was taken after written informed consent was obtained for video recording, and publication in print and online.gait.MRI showed cerebellar atrophy.Sanger sequencing in the sister confirmed the presence of homozygous c.2222G>A;p.Arg741Gln variant.Her dystonia partially improved with levodopa therapy.

Table 3
Table comparing clinical features between our cohort and the MDSgene cohort.
* Information for all 161 patients was not accessible; every variable exhibited missing data, resulting in a fluctuating denominator for each variable.The denominator does not include missing data and is only sum of positive and negative wherever data was available.#Missingdata= 36 (31.3%).&Patientsexhibited multiple behavioral symptoms, including depression, psychosis, and anxiety, either individually or in combination.IQR: Inter quartile range.
[23]er774Iso) in PLA2G6 gene and noted around 70% improvement in UPDRS-III OFF state score at one-year follow-up[23].In the other report by Choi et al. (conference abstract), the authors mention that the siblings carrying compound heterozygous variants (c.359G>A;p.Trp120Ter and c.1742G>A;p.Arg581Gln) in PLA2G6 gene had an excellent response to bilateral STN-DBS